DOES INFECTED SEED SERVE AS INOCULUM SOURCE FOR BOTRYTIS CINEREA INFECTION?

DOES INFECTED SEED SERVE AS INOCULUM SOURCE FOR BOTRYTIS CINEREA INFECTION? *Yahaya S.M. and Ahmed I. Department of Biology, Kano University of Science and Technology, Wudil, P.M.B. 3244Kano State-Nigeria *Author for Correspondence ABSTRACT Isolations were made from lettuce seed and seedlings to determine effect of seed in the transmission of Botrytis cinerea infection in lettuce plant. Commercially purchased lettuce seed Tom Thumb variety was used. Isolation of the pathogen after surface-sterilisation of the seed indicated that the Botrytis cinerea was present within the seed rather than on the surface. It was found that 87% of the seed pathogen detected was B. cinerea and it was able to pass from seed to the resultant seedling, initially appearing in the cotyledon than roots and subsequently in the stems, true leaves or leaf bases and in to the seed. A relationship between level of seed infection by B. cinerea and seedling infection was established (F 1,38 = 51.22, P < 0.001), with higher seed infections resulting in greater seedling infection and leaf rot. This shows that seed is an important source of inoculum for B. cinerea infection. Keywords: Botrytis Cinerea, Dry Spores, Lettuce Seed, Seedling INTRODUCTION Many fungal diseases are seed transmitted where the seed borne infection can spread upward in to the seedling and infect the whole plant body (Stewart and Franicevic, 1994; Barnes and Shaw, 2003; Elias et al., 2010). Although, seed infection can be transmitted in to the seedlings however, it appears seed infection does not always result in seedling infection. This is supported by the work of Elad et al., (2004) who isolated Botrytis fabae, the cause of chocolate spot of Vicia faba from bean seeds. Elad et al., (2004) found that the levels of B. fabae conidia on most infected seeds, which were tested, were too low to cause an aggressive lesion at 15 0 C and the fungus dies without seriously damaging the plant. He concluded that for an aggressive infection to occur seeds would have to carry a high amount of inoculum. Burgess et al., (1997) reported that not all seed infections by endophytic B. cinerea results in seedling infection. The evidence was based on their inability to isolate B. cinerea from sections of surface sterilized asymptomatic epicotyl from seedlings with visible root lesions and healthy seedlings were grown from infected seeds. Stewart and Franicevic (1994) reported that Aspergillus flavus causes seedling infection in young maize plant grown from contaminated seeds. However, it was found that the rate of germination was considerably lower when Aspergillus flavus entered and contaminated the seed. The distribution of the fungus suggests that initially the organism may have followed the meristem of the plant. Ochoa and Ellis (2002) found that Fusarium oxysporum was seed-borne in common naranjilla (Solanum quitoense) and can be transmitted from seed to seedling. Fusarium oxysporum was isolated from seeds and plant parts, which were surface sterilised before plating. In another study Koycu and Ozer (1997) isolated several fungi from onion seeds and seedlings, which were seed-borne, however, only Aspergillus niger and Fusarium oxysporum were transmitted from seed to onion sets. In 1992 Singh et al., found that Botryodiplodia theobromae can be transmitted from seed to seedling in maize. However, they found that heavily infected seeds failed to germinate and were covered with profused growth of pycnidia on their surfaces. While seed with weak to moderate inoculum of Botryodplodia theobromae germinated and their histopathology showed that the fungus readily invades growing seedling from adjoining pericarp, scutellum or closing tissue. They concluded that the fungus grows internally in the seedlings. The fungus was shown to infect the cotyledon immediately after germination and remain as a symptomless infection in the tissue. There have been unpublished reports of high levels of infection of Copyright 2014 Centre for Info Bio Technology (CIBTech) 17

lettuce seed by Botrytis cinerea. However, the relative importance of the seed borne infection in causing the lettuce seedling rot disease has not been clearly established. In addition, the ability of Botrytis to move from infected seed into the seedling tissue was assessed and an attempt was made to correlate levels of seed infection with extent of rot disease at pre and post harvest. Barnes and Shaw (2003) similarly, reported.seed-borne infection of B. cinerea in Primula plant. According to them seed infection lead to seedling infection even when the external inoculum was excluded. Since B. cinerea was isolated from the surface sterilised seeds of lettuce that means the infection was internal. In agreement with the present study the work Sowley (2006) shows that B. cinerea can be transmitted from seed to seedling in lettuce plant. His experiments showed that B. cinerea could be recovered from plants grown from infected seeds under sterile air flow. His outdoor experiments in 2005 showed that B. cinerea could be transmitted from infected plants to seeds, and the same was true for uninoculated plants. This was confirmed by the recovery of B. cinerea from seeds of plants, which were initially grown from apparently uninoculated seeds and later inoculated with the dry B. cinerea spores. However, uncertainties remain with Sowley (2006) experiments due to lack of continuity between his 2005 and 2006 experiments. In 2005 experiments, he used lettuce Little Gem variety and harvested the plants at 1.5 month, but in 2006; he changed the lettuce variety to Tom Thumb and harvested the plants at two months. Furthermore, the environmental condition under which the 2005 experiment was conducted differs from that of 2006 experiments. These differences have put doubt on the results and necessitate the need for the repeat of the experiment using similar lettuce variety under the same environmental condition. The present study hopes to achieve that. Therefore, the study tested two hypotheses. First Infected seed is inoculum source for Botrytis cinerea infection in lettuce. Second that seed infection which grows into the seedling can be transmitted in to the seed. MATERIALS AND METHODS Experimental Plant Seed from a commercially developed lettuce (Tom Thumb variety, Fothergills Seed, Newmarket, UK) was purchased and used for the experiment using the following protocols. Half of the seed was treated with fungicide by soaking the seed in 100ml of the systemic fungicide Shirlan (active ingredient 500g/l Fluazinam, Sygenta Crop Protection UK limited; 0.1g dissolved per litre of water) for 2h, followed by overnight drying (following Shafia, 2009). Prior to sowing in 200 15cm pots, seed sterilisation was carried out to determine if the Botrytis cinerea detected were present on the surface or within the seed. Seeds were blotted dry on sterile filter paper and placed onto 50 plates of Botrytis selective media (25 seeds per plate). Plates were incubated in the dark at 18 O C on Botrytis selective media in a 9cm Petri plate and examined daily for 7 days. Seeds from both fungicide and non-fungicide treated plants were tested. The Petri plates were observed micro- and macroscopically for the presence of B. cinerea. Plant Growth and Seedling Inoculation Seed was sown in 16 trays filled with autoclaved, sterile compost potting mix in the glasshouse facilities of the University of Reading. At the two leaf stage, four trays of seedlings grown from both fungicide and non-fungicide treated seeds were selected and covered with black polyethylene bags. Dried Botrytis cinerea spores collected from a two week-old sporulating culture of B. cinerea in 9cm Petri plates were used for the inoculation. Dry spores were harvested from the Petri plates by tapping gently on an autoclave-sterile piece of aluminium foil. The spores were transferred into 10ml autoclave-sterile syringes (BD Plastipak, UK). The syringes were fitted with 25mm 63/100 23GX1 needles. The needles were inserted into the bag before forcibly delivering the spores into the enclosed area and then left for 24h to facilitate the germination of spores through the build-up of high humidity. An acetate paper on which a graph sheet was photocopied and attached to a glass slides was placed in each tray and examined under a microscope to estimate the number of spores deposited per unit area (an average of 12 spores/mm 2 was found). A week after inoculation, 200 seedlings were transplanted into 15cm pots and maintained under controlled conditions. Copyright 2014 Centre for Info Bio Technology (CIBTech) 18

Isolation of Botrytis Cinerea from Lettuce Seedling To confirm the spread of B. cinerea within the plant body, at intervals of 2, 4, 8 and 12 weeks after sowing 10 seedlings were removed. The roots stems and leaves were surface sterilised for 1 min with 1% NaOCl, followed by three rinses in distilled water. One cm-long sections of secondary root, 1cm diameter leaf discs and hand-cut 1 mm sections of stems were blotted dry on sterile filter paper and segments plated onto Botrytis selective media plates. Plates were incubated in the light at 18 C and examined over a 7-day period. Fungal colonies growing from the section of seedlings were subcultured onto plates of malt extract agar and Botrytis cinerea recorded and identified. Isolation of Botrytis Cinerea from Seed To confirm that B. cinerea had been successfully transferred into the seeds of lettuce plants, ten seeds from the 50 infected and uninfected plants were collected and sterilized by soaking in 20ml of 70% ethanol in a Duran bottle for 30 minutes, and allowed to dry for one hour in a laminar flow cabinet. The seeds from each plant were separately crushed and plated in Botrytis selective medium and incubated (25 seed per plate) at 18 o C for one week. Growth of Botrytis cinerea from each seed was monitored and recorded. Confirmation of the presence of B. cinerea was based on the sporulation of the pathogen and morphological observation of the colonies under the microscope from where BSM stained brown. The colonies isolated were subcultured into MEA in 9cm Petri plates and incubated at 18 o C for one week with alternating UV-A light (12h/day) and dark (12h/day) to allow for the growth of mycelia. The growth of B. cinerea from the seeds to seed was recorded and the data were analysed using ANOVA (Sowley, 2006). Isolation of Botrytis Cinerea from Plant Grown from Infected Seed The seed collected from the inoculated plants which were confirmed to have infection of Botrytis cinerea was grown in controlled environmental room up to the flower stage. The seed was collected from all the 50 plants and tested for the presence of Botrytis cinerea by plating on Botrytis selective media and incubated at 18 o C for one week colony identified micro and macroscopically. Statistical Analysis Factorial analysis of variance was used to evaluate the significance effects of seed infection on the spread of B. cinerea from seed to seedling and back to the next seed (Sokal and Rohlf, 1981; Sowley, 2006). A significant F pr- value in the ANOVA indicates a significant relationship between seed infection and seedling infection P< 0.05. RESULTS AND DISUSSION Results Initial Seed Infection One hundred seeds of lettuce Tom Thumb variety as supplied plated on Botrytis selective media but no infection was detected. Furthermore, no infection was recorded from one hundred seeds of Tom Thumb variety treated with fungicide before plating. However, infection was detected when both fungicides treated and untreated seeds were crushed before plating on Botrytis selective media. There was no significant difference (paired sample t-test, P<0.05)) in the level of Botrytis cinerea isolated from infected and uninfected seed. Isolation of B. cinerea in Lettuce Roots, Stems, and Leaves of Lettuce B. cinerea was detected from root, stems and leaves of plants grown from infected and uninfected seeds. The isolations of B. cinerea at week two and four arose from the root. At week eight and twelve, in addition to the detection of B. cinerea in the root, the fungus was also detected in the stems and leaves. More than half of the plants carried leaf infection of B. cinerea, the incidence of B. cinerea in leaf and root of plants grown from infected seed (75%) was significantly higher than in leaf of plants grown from uninfected seeds (33%) F 1,38 = 61.29, P < 0.001 (Table 1). Copyright 2014 Centre for Info Bio Technology (CIBTech) 19

Table 1: Isolation of B. cinerea from parts of seedlings grown from infected seed Inoculation Plant Part Root Stem Leaves Total Inoculated 90 34 34 158 Uninoculated 82 30 16 128 Isolation of B. cinerea from Seed of Lettuce Plant Grown from Infected Seed Seeds from all the lettuce plant carried the infection of B. cinerea. A total of 364 infections were recorded by plating methods in Botrytis selective media. Seed infection in plants grown from infected seed was statistically significant F 1,38 = 51.22, P < 0.001, while in seeds collected from plant grown from uninfected seed was not significant (Table 2). Table 2: Isolation of Botrytis cinerea from seed of lettuce plants grown from infected seed Infected Uninfected Total Fungicide treated 56 58 112 Non fungicide treated 159 24 183 Discussion Botrytis cinerea was not isolated from water-washed and surface-sterilised source seed. However, when the seeds were crushed before plating infection was detected from both fungicide and non fungicide treated seed. This shows that most of the Botrytis cinerea is present within the seed rather than on the surface. Maude and Presly (1 977a) reported similar results. They found that treatment with Chloros (3% free chlorine) reduced but did not eliminate B. cinerea and they concluded that the majority of the B. cinerea was internal infection rather than surface contamination. This corresponds with the results of isolations made from Primula Polyantha where B. cinerea was the predominant Botrytis species isolated (Barnes and Shaw, 2003). However, it was noted that the proportion of seedlings yielding B. cinerea was less than the proportion of seeds infected with B. cinerea, indicating that not all seed infections result in seedling infections. This observation was supported by the results of Burgess et al., (1997) who observed that seed of chickpea infection did not result in seedling infection. In a related experiment Maude and Presly (1977b) reported similar observations. They found that only 18% of seedlings were infected when seed with a reported 45% infection level was sown. The present study suggests that B. cinerea infects the cotyledon first and subsequently moves down to infect the leaves. This is consistent with the findings of Maude and Presly (1977a and b); Tichelaar (1967); Barnes and Shaw (2003), Shafia (2009); Elias et al., (2010), Tichelaar (1967). In this study, however, a clear relationship between the level of seed infected with B. cinerea and seedling infection was established, with the higher seed infections resulting in greater levels of seedling infection. This evidence suggests that infected seed is an important source of inoculum for infection of lettuce by B. cinerea. However, the present study do not preclude airborne spores of B. cinerea from acting as an additional source of inoculum more over the fact that B. cinerea was not detected in the in some of the original seeds, shows that sometimes it can act as airborne contaminant but spore traps, set out in the field plots, to detect infection shows that B. cinerea were routinely detected. B. cinerea is highly sensitive to the benzimidazoles and dicarboximide group of fungicides. Benomyl (Benlate) and iprodione (Rovral) have been used successfully in the United Kingdom as seed treatments to reduce levels of Botrytis and prevent carry over after harvest (Elias et al., 2010). Therefore given the role of seed borne infection identified by this study, it is recommended that similar seed treatments be used on infected seed lines in an attempt to protect seedlings from Botrytis infection and ultimately reduce the risk of lettuce and other vegetables from rot caused by B. cinerea which may results in wastage, scarcity and high cost of the commodity. Copyright 2014 Centre for Info Bio Technology (CIBTech) 20

ACKNOWLEDGEMENT Thanks to Mark Fellowes, Sue Ramsey and Lee W. Young staffs at School of Biological Science, University of Reading, UK for there kind support and assistance. Also special thanks to Dr. Steve Fuller and Dr. Sally Osborne for proof reading the original version of the manuscript. REFERENCES Barnes SE and Shaw MW (2003). Infection of commercial hybrid Primula seed by Botrytis cinerea and latent disease spread through the plant. Phytopathology 93 573-578. Burgess DR, Bretag TW and Keane PJ (1997). Seed to seedling transmission of B. cinerea chickpea and disinfestation of seed with moist heat. Australian Journal of Experimental Agriculture 37 223-229. Elad Y, Williamson B, Tudzynski P and Delen N (2004). Botrytis spp. and diseases they cause in agricultutal systems- An introduction. In: Botrytis: Biology, Pathology and Control, edited by Elad Y (Springer, Dordrecht, The Netherland) 1-6. Elias SNK, Shaw MW and Dewey FM (2010). Persistent symptomless, systemic and seed-borne infection of lettuce by Botrytis cinerea. European Journal of Plant Pathology 126 61-71. Koycu ND and Ozer N (1997). Determination of seed borne fungi in Onion and Their Transmission to Onion Sets. Phytoparasitica 25 25-31. Maude RB and Presly AH (1977a). Neck rot (Botrytis allii) of bulb onions. I. Seed-borne infection and its relationship to the disease in the onion crop. Annals of Applied Biology 86 163-180. Maude RB and Presly AH (1977b). Neck rot (Botrytis allii) of bulb onions. 11. Seed-borne infection in relationship to the disease in store and the effect of seed treatment. Annals of Applied Biology 86 181-188. Ochoa JB and Ellis MA (2002). Seed transmission of Fusarium oxysporum in common naranjilla (Solanum quitoense) in Ecuador. Plant Health Progress. Shafia A (2009). Latent infection of Botrytis cinerea. PhD Thesis, University of Reading, UK. Singh D and Mathur SB (1992). Systemic infection of maize (Zea mays) seedlings by Botryodiplodia theobromae. Indian Journal of Agricultural Sciences 62 713-716. Sokal RR and Rohlf FJ (1981). Biometry, 2 nd edition (Freeman) San Francisco. Sowley ENK (2006). Epidemiology of Botrytis cinerea, PhD Thesis, University of Reading, UK. Stewart A and Franicevic SC (1994). Infected seed as a source of Inoculum for Botrytis cinerea infection of onion bulb in store. Australian Plant Pathology 23 36-41. Tichelaar GM (1967). Studies on the biology of Botrytis allii on Allium cepa. Netherlands Journal of Plant Pathology 73 157-160. Copyright 2014 Centre for Info Bio Technology (CIBTech) 21